The invention relates to a method for coating antibodies on a substrate for the assay of antigens.
Immunoassays are used to quantify antigens or antibodies by immunochemical means. Generally, a varying quantity of either antigen or antibody is added to a constant amount of the other with the formation of an antigen-antibody complex measured as a function of the varied reactant represented by a standard curve for the varied reactant. The reaction of an unknown amount of the varied reactant can then be referred to the standard curve to obtain the amount of varied reactant which produces a comparable change.
The antibody-antigen complex may form in solution (homogeneous assay) or one of the reactants can be attached to a solid support (heterogeneous assay). The heterogeneous assay generally utilizes a wash step in which uncomplexed material is removed. The present invention pertains only to heterogeneous assays.
In immunoassays, either the antibody or the antigen may be initially affixed to a solid support, such as a plastic surface or beads, to facilitate separation of the antigen-antibody reaction product from unreacted material in immunosorbent assays called solid phase immunoassays. The present invention is concerned with the affixation of antibodies to a solid support.
In solid phase immunoassays, various substances such as fluorochomes, isotopes or enzymes have been used to label antigens or antibodies as a means to detect the antibody-antigen association, for example, fluorescence immunoassay (FIA), radioimmuno-assay (RIA), immunoradiometric assay (IRMA), enzyme immunoassay (EIA), enzyme-linked immunoassay (ELISA), and the biotin-avidin system. Liposomes have also been used as inert reagents to facilitate detection of reaction by incorporating the antigen or antibody into the surface of artificial membranes.
Several methods have been devised for coating a solid phase with an antibody in preparation for solid phase immunoassay. The simplest method is a one step procedure in which a primary antibody is coated in a nonspecific, non-oriented fashion directly onto the surface of a solid phase. This one-step, antibody coated surface is then used to assay for an antigen. (See, e.g., K. J. Catt et al., Nature, 213, 825 ( 1967).
A refinement in the above-described preparation for immunoassays is proper orientation of the primary (capture) antibody for optimal assay sensitivity when the antibody is contacted with antigen. This has been accomplished by use of a secondary antibody which is capable of binding the primary antibody distal to the antigen binding site.
Secondary antibodies may be affinity purified and Fc-specific to a primary antibody. The Fc portion of an immunoglobin (Ig) monomer corresponds to the stem of the Y-shaped Ig molecule and consists of the C-terminal sections of the two heavy chains linked by one or more disulfide bonds. It is the site of complement fixation in complement-fixing antibodies. Fc-specificity enables the primary antibody to bind to the secondary antibody in optimal Fab orientation for immunoassay of the antigen. The Fab portion of an Ig consists of a light chain linked via disulfide bond to the N-terminal part of the heavy chain, i.e. it is one of the two limbs of the Y-shaped Ig molecule. Each Fab portion of an antibody contains a single combining site.
In a multiple coating process, a surface may be first precoated with a secondary antibody which is an anti-antibody to a primary antibody. The pre-coated surface is then coated with the primary antibody to an antigen and the two-coated surface used to assay for the antigen. A method of this type is described, for example, in U.S. Pat. Nos. 4,092,408, and 4,166,844 and by G. M. Sankolli, et al., "Improvement in the Antibody Binding Characteristics of Microtitre Wells by Pretreatment With Anti-IgG Fc Immunoglobin" J. Imm. Meth , 104,191-194 (1987). The coating with secondary and primary antibody may also be simplified by combining the two antibodies into a single-step cocoating process.
When a coating on a solid phase is used in immunoassays, nonspecific binding to unoccupied spaces on the solid surface may interfere with the accuracy, precision or sensitivity of the assay and result in high backgrounds and false read-outs. Blocking agents have been used in a separate coating step to block nonspecific binding sites. Bovine serum, albumin, gelatin, casein, and other substances have been used as blocking agents. This blocking step may be called a postcoat. The postcoating step has traditionally been undertaken after the primary antibody has been applied to the solid surface. In this instance, it can be a 2 step process involving a simple primary antibody coat and a blocking agent-post coat; or a three step process consisting of a secondary antibody precoat, a primary antibody coat, and a blocking agent post coat. If the precoat and coat steps are combined (cocoat), a two step process results with a secondary and primary antibody cocoat in the first step and a blocking agent postcoat in the second step.
A superior coating process using a different sequence of steps has now been discovered.
It is an object of the invention to provide an efficient coating process with a minimum of procedural steps and optimally oriented capture antibody.
It is another object to provide an immunoassay with an increased dynamic range.
An additional object of this invention is to minimize the extent of purification of the primary antibody. Since it is bound directly to the secondary antibody already on the substrate, the primary antibody does not have to compete for binding sites with contaminating proteins.
Another object of this invention is to improve assay response over "traditional" coating methods. The dynamic range is improved over the conventional cocoat-postcoat or precoat-coat-postcoat processes.
Minimized use of primary antibody is yet another object of this invention.